The prevention of cell, tissue or organ transplant rejection and the treatment of autoimmune diseases require either the induction or the restoration of immunological tolerance respectively. In the thymus, tolerance is established through the deletion of auto-reactive T lymphocytes during maturation. In the periphery, tolerance is maintained through several mechanisms including the deletion of auto-reactive T cells, the induction of T cell anergy, or the failure to respond to antigen known as T cell indifference. In the latter case, the autoantigen is present and the T cells are able to respond but the T cells lack the appropriate co-stimulatory signals to respond. Overall, the breakdown of tolerance associated with autoimmune disorders and the lack of tolerance for allografts associated with transplant rejection, are thought to be primarily T-cell mediated immune responses.
CD45 is a transmembrane protein tyrosine phosphatase expressed primarily on cells of hematopoietic origin and is thought to plays a critical role in regulating T-cell activation. CD45 exists as several different isoforms that are generated by the alternate splicing of exons 4-6, also known as A, B and C, from a single primary transcript. A total of 8 isoforms have been described including CD45RO, CD45RA, CD45RB, CD45RC, CD45RAB, CD45RBC, CD45RAC and CD45RABC. These isoforms have different extracellular domains but they have identical cytoplasmic domains including the protein tyrosine phosphatase domains. In humans, naïve helper T-cells and memory T-cells express predominately CD45RA and CD45RO, respectively. The expression of CD45RB appears to be higher on naïve helper T-cells and appears to be down-regulated as the cells become activated.
The literature supports a protective role for anti-CD45RB antibodies in transplant rejection (Zhang et al., Transplant Proc. 27:389 (1995), Lazarovits et al. Nature, 380:717 (1996)). Lazarovits et al. demonstrated the ability of MB23G2, a rat anti-mouse CD45RB antibody, to prevent the rejection of murine renal allografts. This study also demonstrated that MB4B4, another rat anti-mouse CD45RB antibody, was ineffective at preventing renal allograft rejection indicating that not all anti-CD45RB antibodies will show therapeutic efficacy. The antibody MB23G2 has also shown efficacy in promoting the long term survival of pancreatic islet cell allografts (Basadonna et al., PNAS 95:3821 (1998)). The specific mechanism of tolerance induction of the anti-CD45RB antibodies is currently unknown. Tolerance induction by anti-CD45RB antibodies has been correlated with increased tyrosine phosphatase activity (Lazarovits et al., Kidney Int. 55:1303 (1999)). Tolerance may also arise through a specific deletion of alloreactive peripheral blood mononuclear cells or through the induction of T regulatory cells.
Poppema and Lazarovits previously isolated and characterized a murine IgG1 antibody which is directed against human CD45RB, identified as antibody 6G3 (Lazarovits, et al. U.S. Pat. No. 6,024,957, herein incorporated by reference). However, this is a mouse antibody and as such may be highly immunogenic in humans. 6G3, and other antibodies specific for CD45RB, have been shown to prolong renal allograft survival or pancreatic islet cell allografts in cynomolgous monkeys and mice respectively. Functionally, 6G3, and other antibodies specific for CD45RB, will downregulate the expression of CD45RB and L-selectin on peripheral blood mononuclear cells. Interestingly, MB23G2, but not MB4B4, also down-regulates the expression of CD45RB on peripheral blood mononuclear cells supporting the notion that CD45RB down-regulation could be a surrogate marker to identify other anti-CD45RB antibodies capable of transplant protection (Basadonna et al., PNAS 95:3821 (1998)).
Lazarovits, et al. U.S. Pat. No. 6,106,834 identified a method of identifying ligands (antibodies) specific for CD45RB, CD45RO using an immunoaffinity column (ie: with MT3 or 6G3 antibodies). The immunoaffinity column is used to identify an antigen-containing binding partner. Then the binding partner is used to select cross-reactive ligands. However, specific antobodies which recognize the CD45RB isotype were not identified.
Lazarovits, et al. U.S. Pat. No. 6,024,957 describe a method of humanizing the previously identified MT3, or 6G3 antibodies which would be less likely to be recognized by the immune system as foreign. However, these antibodies are still not fully human and may induce a reaction when introduced into the human immune system.
Thus, anti-CD45RB-specific antibodies which are specific for a variety of antigenic determinants of CD45RB are needed which do not recognize the other isoforms of the CD45 antigen. To be of use in humans, these antibodies should be fully human and display similar functional characteristics as 6G3. In addition, large scale concentrations of the antibodies should be producible with a minimum of labor.